Biomedical subjects

Michael D Leavell

Publications and source records attributed to Michael D Leavell.

12 recordsLinked to original sources

PubMed2006

Fatty acid analysis tool (FAAT): An FT-ICR MS lipid analysis algorithm.

Electrospray ionization mass spectrometry is becoming an established tool for the investigation of lipids. As the methods for lipid analysis become more mature and their throughput increases, computer algorithms for the interpretation of such data will become a necessity. Toward this end, an algorithm dedicated to the analysis of Fourier transform mass spectral data from lipid extracts has been developed. The algorithm, Fatty Acid Analysis Tool, termed FAAT, has been successfully used to investigate complex lipid extracts containing thousands of components, from various species of mycobacteria including M. tuberculosis and M. abscessus. FAAT is rapid, generally taking tens of seconds to interpret multiple spectra, and accessible to most users as it is implemented in Microsoft Excel Visual Basic Software. In the reduction of data, FAAT begins by scaling spectra (i.e., to account for dilution factors), identifying monoisotopic ions, and assigning isotope packets. Unique features of FAAT include the following: (1) overlapping saturated and unsaturated lipid species can be distinguished, (2) known ions are assigned from a user-defined library including species that possess methylene heterogeneity, (3) and isotopic shifts from stable isotope labeling experiments are identified and assigned (up to a user-defined maximum). In addition, abundance differences between samples grown under normal and stressed conditions can be determined. In the analysis of mycobacterial lipid extracts, FAAT has successfully identified isotopic shifts from incorporation of 15N in M. abscessus. Additionally, FAAT has been used to successfully determine differences in lipid abundances between M. tuberculosis wild-type and mutant strains.

Algorithms↗

PubMed2006

A sulfated metabolite produced by stf3 negatively regulates the virulence of Mycobacterium tuberculosis.

Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells. Mycobacterium tuberculosis, the causative agent of tuberculosis, produces sulfated molecules that have eluded functional characterization for decades. We demonstrate here that a previously uncharacterized sulfated molecule, termed S881, is localized to the outer envelope of M. tuberculosis and negatively regulates the virulence of the organism in two mouse infection models. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate and a previously uncharacterized sulfotransferase, stf3. These findings extend the known functions of sulfated molecules as general modulators of cell-cell interactions to include those between a bacterium and a human host.

Animals↗

PubMed2005

Unambiguous assignment of intramolecular chemical cross-links in modified mammalian membrane proteins by Fourier transform-tandem mass spectrometry.

Fourier transform tandem mass spectrometry (FT-MS/MS) can be used to unambiguously assign intramolecular chemical cross-links to specific amino acid residues even when two or more possible cross-linking sites are adjacent in the cross-linked protein. Bovine rhodopsin (Rho) in its dark-adapted state was intramolecularly cross-linked with lysine-cysteine (K-C) or lysine-lysine (K-K) cross-linkers to obtain interatomic distance information. Large, multiply charged, cross-linked peptide ions containing adjacent lysines, corresponding to Rho(50-86) (K(66) or K(67)) cross-linked to Rho(310-317) (C(316)) or Rho(318-348) (K(325) or K(339)), were fragmented by collision-induced dissociation (CID), infrared multiphoton dissociation (IRMPD), and electron capture dissociation (ECD). Complementary sequence-specific information was obtained by combining cross-link assignments; however, only ECD revealed full palmitoylation of adjacent cysteines (C(322) and C(323)) and cross-linking of K(67) (and not K(66)) to C(316), K(325), and K(339). ECD spectra contained crucial c- and z-ions resulting from cleavage of the bond between K(66) and K(67). To our knowledge, this work also presents the first demonstration that ECD can be used to characterize S-linked fatty acid acylation on cysteines. The comprehensive fragmentation of large peptides by CID, IRMPD, and particularly ECD, in conjunction with the high resolution and mass accuracy of FT-MS/MS, is shown to be a valuable means of characterizing mammalian membrane proteins with both chemical and posttranslational modifications.

Amino Acid Sequence↗

PubMed2004

Screening and identification of acidic carbohydrates in bovine colostrum by using ion/molecule reactions and Fourier transform ion cyclotron resonance mass spectrometry: specificity toward phosphorylated complexes.

A screening method was developed for the identification of acidic saccharides from biological mixtures utilizing gas-phase derivatization and mass spectrometry. Phosphorylated compounds were differentiated from other acidic species by exploiting the selective reactivity of chlorotrimethylsilane with the phosphate ions (phosphorylated compounds shift by 72 Da, allowing rapid compound detection). A 13-component mock mixture was used to demonstrate the viability of the method, and a detection limit of 600 nM (30 fmol) was determined. This method was applied to the identification of acidic compounds from bovine colostrum. To further verify the selectivity of the ion/molecule reaction, exact mass measurements were used to determine the elemental composition of 14 compounds. Eight novel acidic carbohydrate species were observed in bovine colostrum, six of which have never been reported previously in milks. Tandem mass spectrometric experiments allowed compound characterization for two of these components.

Animals↗

PubMed2004

Strategy for selective chemical cross-linking of tyrosine and lysine residues.

Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin beta-chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminus-tyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH.

Angiotensin I↗

PubMed2003

MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence.

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is unique among bacterial pathogens in that it displays a wide array of complex lipids and lipoglycans on its cell surface. One of the more remarkable lipids is a sulfated glycolipid, termed sulfolipid-1 (SL-1), which is thought to mediate specific host-pathogen interactions during infection. However, a direct role for SL-1 in M. tuberculosis virulence has not been established. Here we show that MmpL8, a member of a large family of predicted lipid transporters in M. tuberculosis, is required for SL-1 production. The accumulation of an SL-1 precursor, termed SL(1278), in mmpL8 mutant cells indicates that MmpL8 is necessary for an intermediate step in the SL-1 biosynthesis pathway. We use a novel fractionation procedure to demonstrate that SL-1 is present on the cell surface, whereas SL(1278) is found exclusively in more internal layers. Importantly, we show that mmpL8 mutants are attenuated for growth in a mouse model of tuberculosis. However, SL-1 per se is not required for establishing infection as pks2 mutants, which are defective in an earlier step in SL-1 biosynthesis, have no obvious growth defect. Thus, we hypothesize that either MmpL8 transports molecules in addition to SL-1 that mediate host-pathogen interactions or the accumulation of SL(1278) in mmpL8 mutant cells interferes with other pathways required for growth during the early stages of infection.

Bacterial Proteins↗

PubMed2003

Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers. an FT-ICR approach.

A rapid and accurate method of quantifying positional isomeric mixtures of phosphorylated hexose and N-acetylhexosamine monosacchrides by using gas-phase ion/molecule reactions coupled with FT-ICR mass spectrometry is described. Trimethyl borate, the reagent gas, reacts readily with the singly charged negative ions of phosphorylated monosaccharides to form two stable product ions corresponding to the loss of one or two neutral molecules of methanol from the original adduct. Product distribution in the ion/molecule reaction spectra differs significantly for isomers phosphorylated in either the 1- or the 6-position. As a result, the percents of total ion current of these product ions for a mixture of the two isomers vary with its composition. In order to determine the percentage of each isomer in an unknown mixture, a multicomponent quantification method is utilized in which the percents of total ion current of the two product ions for each pure monosaccharide phosphate and the mixture are used in a two-equation, two-unknown system. The applicability of this method is demonstrated by successfully quantifying mock mixtures of four different isomeric pairs: Glucose-1-phosphate and glucose-6-phosphate; mannose-1-phosphate and mannose-6-phosphate; galactose-1-phosphate and galactose-6-phosphate; N-acetylglucosamine-1-phosphate and N-acetylglucosamine-6-phosphate. The effects of mixture concentrations and ion/molecule reaction conditions on the quantification are also discussed. Our results demonstrate that this assay is a fast, sensitive, and robust method to quantify isomeric mixtures of phosphorylated monosaccharides.

Fourier Analysis↗

PubMed2002

Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach.

The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell-cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SO 4 2-). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.

Cysteine↗

PubMed2002

Analysis of phosphate position in hexose monosaccharides using ion-molecule reactions and SORI-CID on an FT-ICR mass spectrometer.

Through the use of ion-molecule reactions and SORI-CID, the phosphate position in hexose phosphate monosaccharides has been determined in the negative ion mode. Trimethyl borate was used as a reagent gas and was found to react readily with the phosphorylated hexose monosaccharides. After reaction of the reagent gas with the hexose phosphate, ion activation of the precursor by SORI-CID yielded different MS/MS spectra. Different diagnostic ions were generated for the two isomers, thus enabling differentiation and linkage position determination of the phosphate moiety.

Fourier Analysis↗

PubMed2002

Solvent effects in tandem mass spectrometry: mechanistic studies indicating how a change in solvent conditions and pH can dramatically alter CID spectra.

Dramatically different CID (collision-induced dissociation) spectra are obtained when the complex [Zn(dien-glucose)](+) is electrosprayed from acidic and basic solutions. To understand this peculiar phenomenon, an in-depth mechanistic study was performed on one of the product ions that is present when the initial complex is diluted in basic solution but absent when the complex is diluted with acidic solution. On the basis of the results of this study, the differences in the CID spectra can be rationalized by the fact that the complex electrosprayed from basic solution was kinetically trapped, with the deprotonation site distal from the metal center. Under acidic conditions, the deprotonation site is at a hydroxyl group coordinated to the metal ion. A variety of experiments support this hypothesis. The studies herein underscore the importance of using identical solvent conditions when comparing sets of CID spectra. The data also highlight a very interesting phenomenon involving deprotonation of a hydroxyl group, which was several atoms away from the Zn(II) metal center.

Glucose↗

PubMed2002

Conformational studies of Zn-ligand-hexose diastereomers using ion mobility measurements and density functional theory calculations.

Ion mobility studies and density functional theory calculations were used to study the structures of [Zn/diethylenetriamine/Hexose/Cl]+ complexes in an effort to probe differences in the three-dimensional conformations. This information allows us to gain insight into the structure of these complexes before collisional activation, which is the first step in understanding the stereoselective dissociations observed under collisionally activated conditions. The collision cross sections obtained from the ion mobility measurements showed that the mannose structure is more compact than the galactose and glucose complexes, respectively. Using density functional theory, candidate structures for each of the experimentally observed complexes were generated. Two criteria were used to determine the most likely structures of these complexes before activation: (1) The allowed relative energies of the molecules (between 0-90 kJ/mol) and (2) collision cross section agreement (within 2%) between the theoretically determined structures and the experimentally determined cross section. It was found that the identity of the monosaccharide made a difference in the overall conformation of the metal-ligand-monosaccharide complex. For glucose and galactose, metal coordination to O(6) was found to be favorable, with the monosaccharide occupying the 4C1 chair conformation, while for mannose, O(2) metal coordination was found with the monosaccharide in a B3,0 conformation. Coordination numbers varied between four and six for the Zn(II) metal centers. Given these results, it appears that the stereochemistry of the monosaccharide influences the conformation and metal coordination sites of the Zn(II)/monosaccharide/dien complex. These differences may influence the dissociation products observed under collisionally activated conditions.

Algorithms↗

PubMed2002

Mass spectrometric strategy for the characterization of lipooligosaccharides from Neisseria gonorrhoeae 302 using FTICR.

The lipooligosaccharides (LOS) of Neisseria gonorrhoeae 302 were profiled using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Using techniques developed in this laboratory, the topology and some of the linkages of the LOS were determined. Mass spectrometric analysis in the negative ion mode yielded a glycoform of the composition: Hex3 Hep2 Hxn1 PEA1 KDO2 DPLA. The composition was confirmed through exact mass measurements, which showed only a 2 ppm error between the exact mass and theoretical mass. Although the core structure has been postulated previously, the positioning of the three hexose moieties were in question for this particular strain of N. gonorrhoeae. Topology assignment was performed through collision-induced dissociation analysis of the O-deacylated glycoform in the negative ion mode followed by submission to the saccharide topology analysis tool (STAT) computer program, which confirmed the topology assignment. It was found that the three hexoses were added to the Hep[I] of the conserved core of N. gonorrhoeae in a linear fashion, while Hep[II] remains unbranched. Linkage position analysis was performed through application of a mild acid hydrolysis technique followed by collision-induced dissociation of the sodiated precursor ions, yielding a 1 --> 4 linkage between the terminating and penultimate hexoses.

Antigens, Surface↗